1. Field of the Invention
The present invention relates to cell lines, cell cultures, compositions and cell culture media. More specifically, the present invention relates to methods to develop new cell lines and to increase cell culture population doublings of finite life span human cells. The present invention also relates to using the described cell cultures and methods for assessment of factors influencing aging and/or carcinogenesis.
2. Related Art
Human mammary epithelial cells (HMEC), the cell type from which breast cancers originate, normally express a finite reproductive potential in culture. This cellular senescence can be enforced by the repression of telomerase, the enzymatic activity that allows cells to maintain the integrity of the chromosome ends, as well as by a stress-induced mechanism (stasis), and by exposure to overexpressed oncogenes (OIS: oncogene-induced senescence). Telomerase expression is found in cancer cells; the resultant immortality allows cancers to accumulate the errors necessary for malignancy. Thus, telomerase repression is thought to be a tumor suppressor mechanism. Low telomerase expression may be present in some finite HMEC, and this telomerase activity may perform functions other than maintaining chromosomal ends.
For a normal, finite life span human breast cell to become malignant, multiple aberrations in pathways governing growth control and invasive potential need to accumulate. The errors seen in human breast cancers commonly involve defects in the retinoblastoma (RB) pathway, and almost always include reactivation of telomerase activity. The immortal potential conferred by telomerase is thought to be crucial for error accumulation, and is due to the ability of telomerase to maintain stable telomere lengths via de novo addition of telomeric repeat sequences. Expression of telomerase activity, by itself, is not thought to confer malignancy-associated properties (Morales, C. P. et al. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nature Gen., 21: 115-118, 1999; Jiang, W. R. et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nature Gen., 21: 111-114, 1999; Wei, S. et al. Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts. Cancer Res., 59: 1539-1543, 1999.), however, a variety of recent studies suggest that telomerase may perform non-telomere length maintenance functions that affect cell behavior (Stampfer, M. et al. Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4(−) human mammary epithelial cells. Proc. Natl. Acad. Sci., USA., 98: 4498-4503, 2001; González-Suárez, E. et al. Increased epidermal tumors and increased wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratonocytes. EMBO J., 20: 2619-2630, 2001; Oh, H. et al. Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci USA, 98: 10308-11033, 2001; Lu, C. L. et al. Telomerase protects developing neurons against DNA damage-induced cell death. Dev. Brain Res., 131: 167-171, 2001; Blasco, M. A. Telomerase beyond telomeres. Nat Rev Cancer, 2: 627-632, 2002; Smith, L. L. et al. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol, 5: 474-479, 2003; Masutomi, K. et al. Telomerase maintains telomere structure in normal human cells. Cell, 114: 241-253, 2003.).
Experimental examination of certain hypotheses have previously been very difficult to perform in normal finite life span human mammary epithelial cells, or other normal finite life span human epithelial cells, due to the rapid onset of stasis once normal human epithelial cells are placed in culture. A consequence of the limited pre-stasis growth has been that studies requiring large numbers of cells, or large standardized cell batches, were difficult to perform. Most large-scale studies using cultured finite life span HMEC have employed cell cultures originally developed and termed post-selection HMEC, i.e., cells that had spontaneously silenced p16 expression, thus overcoming stasis (Brenner, A. J. et al. Increased p16INK4a expression with onset of senescence of human mammary epithelial cells and extended growth capacity with inactivation. Oncogene, 17: 199-205, 1998; Hammond, S. L. et al. Serum-free growth of human mammary epithelial cells: Rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc. Natl. Acad. Sci. USA, 81: 5435-5439, 1984.).